U.S. patent number 10,788,484 [Application Number 16/022,259] was granted by the patent office on 2020-09-29 for apparatus and method for absolute quantification of biomarkers for solid tumor diagnosis.
This patent grant is currently assigned to QUANTICISION DIAGNOSTICS INC.. The grantee listed for this patent is QUANTICISION DIAGNOSTICS INC.. Invention is credited to Jiandi Zhang.
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United States Patent |
10,788,484 |
Zhang |
September 29, 2020 |
Apparatus and method for absolute quantification of biomarkers for
solid tumor diagnosis
Abstract
Provided is a method for quantitative analysis of a sample. The
method includes steps of (a) providing a singular marker
representative of one or more features of the sample, the sample
comprising a population of individual units of the marker; (b)
measuring the marker with dot blot analysis, wherein the
quantitation result is an absolute amount of the marker's
population of individual units in the sample, normalized by the
sample volume or by the sample weight; and (c) obtaining an
objective determination of the one or more features of the sample
based on the quantitation result of the marker. Also disclosed is a
reference database and a method to use the reference database for
diagnosing cancer in a patient.
Inventors: |
Zhang; Jiandi (Chapel Hill,
NC) |
Applicant: |
Name |
City |
State |
Country |
Type |
QUANTICISION DIAGNOSTICS INC. |
Chapel Hill |
NC |
US |
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Assignee: |
QUANTICISION DIAGNOSTICS INC.
(Chapel Hill, NC)
|
Family
ID: |
1000005082584 |
Appl.
No.: |
16/022,259 |
Filed: |
June 28, 2018 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20190004037 A1 |
Jan 3, 2019 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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62526425 |
Jun 29, 2017 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N
33/574 (20130101); G01N 33/54306 (20130101); G01N
33/57484 (20130101); G01N 33/6893 (20130101); G01N
2800/52 (20130101); G16H 50/20 (20180101) |
Current International
Class: |
G01N
33/543 (20060101); G01N 33/68 (20060101); G01N
33/574 (20060101); G16H 50/20 (20180101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
Other References
Ura et al. (Int. J. Cancer 1992 50:57-63). (Year: 1992). cited by
examiner .
Tan et al. (PLoSOne 2011 6:e18764). (Year: 2011). cited by examiner
.
CoAA (Sui et al. Oncogene 2007 26:822-835) (Year: 2007). cited by
examiner .
Guillemin et al. "Validation of a Dot-Blot quantitative technique
for large scale analysis of beef tenderness biomarkers," J Physiol
Pharmacol, Oct. 1, 2009 (Oct. 1, 2009), vol. 60, Suppl. 3, pp.
91-97. cited by applicant .
Venugopal et al. "Quantitative proteomics for identifying
biomarkers for Rabies," Clin Proteomics, Mar. 22, 2013 (Mar. 22,
2013), vol. 10, No. 3, pp. 1-13. cited by applicant .
Tian et al. "Quantitative dot blot analysis (QDB), a versatile high
throughput immunoblot method," Oncotarget, Apr. 19, 2017 (Apr. 19,
2017), vol. 8, No. 35, pp. 58553-58562. cited by applicant .
International Searching Authority/US, "PCT International Search
Report" for PCT/US2018/040075, dated Sep. 13, 2018. cited by
applicant.
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Primary Examiner: Cheu; Changhwa J
Attorney, Agent or Firm: Novick, Kim & Lee, PLLC Xue;
Allen
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a U.S. nonprovisional patent application which
claims the benefit of provisional application No. 62/526,425, filed
on Jun. 29, 2017, the disclosure of which is hereby incorporated by
reference. Further, U.S. application Ser. No. 14/721,205, filed on
May 26, 2015, and U.S. application Ser. No. 15/433,586, filed on
Feb. 15, 2017 (now U.S. Pat. No. 10,225,544), are also hereby
incorporated by reference in their entireties for all purposes.
Claims
The invention claimed is:
1. A method for quantitative analysis of a sample, comprising: (a)
providing a singular marker representative of one or more features
of the sample, the sample being a formalin-fixed paraffin-embedded
(FFPE) tissue sample having a population of individual units of the
marker; (b) extracting markers from the tissue sample and measuring
the marker with quantitative dot blot analysis (QDB), wherein the
quantitation result is an absolute amount of the marker's
population of individual units in the sample, normalized by the
protein weight, and the marker standard curve is established to
convert signals captured into absolute marker level; and (c)
obtaining a determination of the one or more features of the sample
based on the absolute quantitation result of the marker.
2. The method of claim 1, wherein the step (b) comprises: (b1)
incubating the sample with a solution comprising a binding agent,
wherein: the binding agent is capable of specifically binding to an
individual unit of the marker, and the binding agent is capable of
being quantified by the dot blot analysis; and (b2) measuring the
marker through QDB analysis of the binding agent.
3. The method of claim 2, wherein the marker is a protein marker
and the binding agent is an antibody.
4. The method of claim 3, wherein the antibody is an analyte
specific regent (ASR) antibody.
5. The method of claim 3, wherein the antibody is an in vitro
diagnostics (IVD) antibody.
6. The method of claim 3, wherein the antibody is capable of being
assayed in immunohistochemistry.
7. The method of claim 1, wherein the sample is a biopsy tissue
from a subject.
8. The method of claim 7, wherein the subject is a cancer
patient.
9. The method of claim 8, further comprising (d) evaluating the
patient based on the quantitation result of the marker.
10. The method of claim 9, wherein, in step (d), evaluating the
patient comprises diagnosis and prognosis of a cancer for the
patient.
11. The method of claim 10, wherein the diagnosis and prognosis of
a cancer comprises disease-free survival, overall survival, hazard
ratio, or treatment prediction for the cancer.
12. A method for diagnosing cancer in a patient, comprising: (a)
obtaining a biopsy sample from the patient; (b) measuring a marker
in the biopsy sample with QDB analysis, wherein the measured result
is an absolute amount of the marker in the biopsy sample normalized
by protein weight and the marker standard curve is established to
convert signals captured into absolute level; (c) comparing the
quantitation result of the marker with that of a reference profile
stored in a reference database; wherein the reference profile
database is obtained by measuring the biomarker from samples of a
known cancer diagnosis using the same quantitative measurement of
QDB analysis of step (b); and (d) identifying a reference profile
in the reference database that has the best match and administering
therapeutics to said patient.
13. The method of claim 12, wherein the cancer is a solid tumor
cancer.
Description
FIELD OF THE INVENTION
The present disclosure relates to methods and apparatus for
quantification of biomarker at tissue level for diagnostics purpose
using immunoblot method. Specifically, this disclosure provides
methods and apparatus to measure the expression level of a
biomarker using quantitative dot blot analysis (QDB), and when
combined with a reference database, to provide diagnosis and
prognosis for solid tumors.
BACKGROUND
Protein analysis is the basis of modern biological research. It
centers on antigen-antibody interaction to measure levels of
antigen of interest under various medical or experimental
conditions. An antigen by definition is a foreign molecule that
triggers the production of an antibody by the immune system when
introduced into the body. The high specificity of the antibody
against a specific antigen makes it a powerful tool in clinical,
pharmaceutical and biomedical research.
An antigen includes, but not limited to a chemical compound, a
peptide, a protein, an RNA, a DNA, a cell (proteins released in
situ), or a virus particle (proteins released in situ). The
molecule of antigen, as a whole or in part, may be introduced into
a host animal, such as a donkey, a goat, or a rabbit to generate a
large quantity of antibody against the introduced antigen of
interest. Furthermore, the introduced antigen, or part of the
antigen, may have more than one epitopes, thus may generate a
corresponding number of antibodies against the antigen of
interest.
A typical immunodetection process has three major steps. The first
step is sample application, in which prepared samples containing an
antigen of interest is first bound to a solid phase, such as
nitrocellulose or PVDF membrane, multi-well plate with protein
binding capacity, glass slide in immunohistochemistry analysis
(IHC) or membrane at QDB plate, Zhang et al. "Quantitative dot blot
analysis (QDB), a versatile high throughput immunoblot method"
Oncotarget 2017 Vol. 8, page 58553-58562, which is incorporated by
reference herein in its entirety. The second step is to form and
label the antigen-antibody complex (i.e., immunocomplex) of
interest. This step involves the sub-steps of blocking, incubation
and washing. In the blocking sub-step, non-specific protein binding
sites on the membrane are blocked using a blocking buffer to shield
them from non-specific protein. In some case of IHC, slice on the
glass slide needs to be further processed (de-paraffined and
antigen retrieval) before the blocking buffer is applied. After
blocking, the membrane is incubated in the incubation step with
antibody against the antigen of interest to form membrane-bound
antigen-antibody complex. The unbound antibodies are washed away.
The antibody used herein is often commercially available as a
pre-labelled antibody. One may also perform the labeling sub-step
on site. In either case, the antibody shall be labeled, either
directly with a reporter, e.g., a reporter enzyme, or indirectly
labelled using a secondary antibody conjugated with a reporter.
The third step is detection. Signals emitted by the reporter enzyme
are detected and recorded, which yield information related to the
quantity or quality of the immunocomplex bound on the membrane.
Different methods of labeling the antibody in turn necessitate
different corresponding detection methods. For example, the third
step of detection may be a color reaction by visual inspection or
chemiluminescence signals detectable through scanner, X-ray film or
microplate reader, etc. The antibody may also be
fluorescence-labeled and detected through scanner
Multiple techniques of protein analysis are variations of this
typical immunodetection process. These techniques include, but not
limited to, Western blot analysis, Dot blot analysis,
immunohistochemistry (IHC), Enzyme-linked immunosorbent assay
(ELISA), reverse phase protein microarray (RPPM) and newly
developed Quantitative Dot Blot analysis (QDB) and Zestern
analysis.
In immunohistochemical analysis, the tissue is first processed
either as paraffin-embedded block or as a frozen tissue, and is
further sliced at certain thickness before the tissue slice is
placed on a slide. Next, the slide is going through a typical
immunodetection process to form immunocomplex between antigen of
interest and detection antibody on the slide. The pre-labeled
antibody is used to allow detection of antigen of interest on the
slide by a pathologist with the aid of microscope. For slice from
paraffin-embedded block, the slice needs to go through further
steps to facilitate antigen-antibody interaction. These steps may
include de-paraffin of the slice, and antigen retrieval step to
expose the antigen for antigen-antibody interaction.
In clinical practice, IHC is widely used to detect any changes at
protein level of a biomarker for diagnostic or prognostic purpose.
A typical IHC report of a biomarker either expresses as "+" or "-",
or it is further categorized as "0, 1+, 2+, 3+". For example, the
expression level of one commonly used biomarker for breast cancer
diagnosis, Human epidermal growth factor receptor 2 (Her2), is
assessed using IHC to determine if Her2-dependent therapy should be
included in the treatment plan. The IHC results are categorized
into four groups: 0 and 1+, 2+, and 3+. 0 and 1+ group is
considered as negative, 3+ is considered as positive while 2+ is
considered as equivocal.
Attempts trying to unite the intensity of staining with the
percentage of cells stained lead to scoring system including H
score (or H index) developed to provide more informative detail
about IHC analysis. In other cases, the intensity of the staining,
for example, strong or weak and the percentages of the cells
stained are both provided. For example, the results can be reported
as (percentage, intensity). However, all these results are
semi-quantitative in nature.
Although IHC provides valuable morphological information, and
numerous efforts have been invested to standardize the analytical
procedure, its result is still qualitative, affected by various
factors including the process of treatment, the field of view and
inter-observer variability. It is also rather challenging to adapt
this technique into a high throughput format.
The uncertainty associated with IHC analysis consequently leads to
significantly increased error in diagnosis. In fact, in Her2's
case, there are as much as 30% inconsistency between IHC with
Fluorescence In Situ Hybridization technique (FISH).
The categorized results of IHC analysis also makes it difficult for
further data analysis. For example, while there are significant
differences among individual patients with positive results, they
are all considered in the same category in clinical practice. Thus,
results from IHC analysis are unable to be used for extensive data
analysis to provide more accurate, more predictive diagnosis or
prognosis as we expected.
There have been several attempts to measure quantitatively the
biomarker at tissue level with success. For example, Hermark assay,
based on the proximity assay, was able to quantify Her2 levels at
breast cancer tissues.sup.4. However, this technique measures the
relative Her2 level among breast cancer patients.
In the meantime, the absolute Her2 level was achieved with SRM-MS
technique. Using an isotope labeled protein standard, the absolute
Her2 levels were measured, and a cutoff of 740 amole/.mu.g was
proposed based on the Receiving Operative Characteristics (ROC)
analysis.
Nonetheless, these methods are complicated and labor intensive, not
suitable for high throughput analysis of a large number of samples.
The complex process also leads to significantly increased operating
cost and efforts.
The recently developed QDB method, on the other hand, has been
demonstrated suitable to process complex lysates prepared either
from cells or tissues in a high throughput format. Introduction of
a protein standard, either in the form of a recombinant protein, or
a purified protein, conveniently translates this method into an
absolute quantitative assay to measure the absolute content of a
specific protein at cellular or tissue level.
In this invention, the effort was made to adopt QDB assay in
measuring the expression level of a biomarker at tissue level for
clinical use. In one application of current invention, the IHC
assays currently available for diagnostics purpose can be converted
into absolute quantitation assays with clear advantages of
accurate, quantitative, objective, high throughput, and more
comprehensive to aid clinical diagnosis or prognosis. The absolute
values from QDB analyses can be further processed in large scale
for data analysis to provide more predictive and accurate diagnosis
and prognosis.
SUMMARY OF THE INVENTION
The present invention provides method to quantify the expression
levels of biomarkers at tissue levels. The evaluation of biomarkers
is quantitatively measured instead of categorized as in current
prevailing methods.
Consequently, the present invention provides a continuous result to
replace the current prevailing discrete result. Thus, the present
invention opens more opportunities than the current prevailing
method of IHC and FISH.
One aspect of the present invention relates to a method for
quantitative analysis of a sample. The method includes steps of (a)
providing a singular marker representative of one or more features
of the sample, which contains a population of individual units of
the marker; (b) measuring the marker with dot blot analysis,
wherein the quantitation result is an absolute amount of the
marker's population of individual units in the sample, normalized
by the sample volume or by the sample weight; and (c) obtaining an
objective determination of the one or more features of the sample
based on the quantitation result of the marker.
The sample can be a tissue from a subject. In one embodiment of
current invention, the tissue refers to a biopsy tissue. In another
embodiment of current invention, the tissue refers to a frozen
tissue. Yet in another embodiment of current invention, the tissue
refers to a Formalin Fixed specimen, and yet in another embodiment
of current invention, the tissue refers to a Formalin Fixed
Paraffin Embedded specimen (FFPE specimen).
The subject can be a patient. Specifically, the subject can be a
cancer patient.
On the other hand, the step (b) of the above method can include
measuring the marker with quantitative dot blot (QDB) analysis.
More specifically, the step (b) can include: (b1) incubating the
sample with a solution containing a binding agent, which is capable
of specifically binding to an individual unit of the marker and is
capable of being quantified by the dot blot analysis; and (b2)
measuring the marker through QDB analysis of the binding agent.
The binding agent can be capable of being assayed based on
antibody-antigen interaction including immunohistochemistry.
Moreover, the binding agent can be capable of being assayed in flow
cytometry.
Preferably, the marker is a protein marker and the binding agent is
an antibody. Further, the antibody can be an analyte specific
regent (ASR) antibody, and it can also be an in vitro diagnostics
(IVD) antibody.
Additionally, the above-described method further includes step (d)
of evaluating the patient based on the quantitative result of the
marker. Particularly, step (d) includes diagnosis and prognosis of
a cancer for the patient. Examples of diagnosis and prognosis of a
cancer for the patient include disease-free survival, overall
survival, Hazard Ratio (HR), or treatment prediction for the
cancer.
The quantitatively measured biomarker level disclosed in this
invention can be categorized based on mathematical analysis of
available information to facilitate the comparison from this
invention to the current prevailing method, and to help better
explore the clinical significance of the absolute level of a
biomarker.
One embodiment of current invention is through immunoblot analysis.
The expression level of a biomarker can be measured using an
antibody-based method.
By defining the linear range of the antibody-based method, the
absolute level of a biomarker can be measured with a protein
standard in the same measurement.
In one embodiment of current invention, dot blot method can be used
to achieve the absolute level of a biomarker at tissue.
Purified or recombinant protein is used to establish a protein
standard curve using image-based dot blot analysis. The image is
converted into number through image capture and analysis.
The captured signal is used to establish a protein standard
curve.
Total protein extracted from FFPE block can be used in dot blot
analysis, and the captured signal can be converted into absolute
biomarker level using the referenced standard curved described
above.
In one embodiment of current invention, the Quantitative Dot Blot
analysis (QDB) method can be used to achieve the absolute level of
a biomarker at tissue level.
The protein expression level of a biomarker can be measured
relatively through immunoblot assay including QDB method in the
absence of a protein standard. As long as the process of analysis
is well controlled (e.g., with the positive and negative controls),
it provides informative result for reference.
In QDB analysis, the absolute level of a biomarker can be measured
using a validated detection antibody. There is no limitation of
methods to validate the detection antibody. The antibody can be
validated through Western blot analysis, or it can be validated
based on prior experience. In one embodiment of current invention,
a well-accepted antibody for immunohistochemistry can be used
directly in QDB analysis. For example, ASR or IVD antibodies can be
used in QDB analysis. These antibodies include, but not limited to,
EP3 and 4B5 clones for HER2 protein, MIB1 for ki67 and SP1 for
Estrogen Receptor (ER). Likewise, a detection antibody for other
antibody-based assay, including, but not limiting, flow cytometry,
can be used in QDB analysis.
A detection antibody (antibody A) can be validated through
comparative study with an existing detection antibody (antibody B).
When more than one sample are analyzed with both antibody A and
antibody B with similar result, antibody A can be considered
validated
Sample for QDB analysis can be prepared from frozen tissue or from
a formalin-fixed tissue or from a formalin-fixed-paraffin-embedded
tissue block (FFPE block). It can also be prepared from tissues
preserved in other format as long as the antigen-antibody
interaction is allows to happen with the sample.
The absolute level of a biomarker from more than one sample can be
combined with relevant clinical information to setup a protein
database (AKA, QDB database). Mathematical analysis of QDB database
provides information for medical use. For example, the putative
association between absolute level of a biomarker and the disease
free survival (DFS) can be explored to provide predictive clinical
prognosis for a patient.
These and other embodiments of the present invention rely at least
in part upon the finding that the absolute nature of the QDB
analysis allows for combining results from more than one source for
analysis. The continued addition of new information from QDB
analysis allows growing of a QDB database. Information obtained
through analysis of a QDB database provides diagnosis or prognosis
for a patient.
Protein can be extracted in non-denatured form, or it can be
further denatured through heating and/or reducing reagent. The
non-denatured form should be taken in their broadest context. It
can be in native form; or it may refer a state where the antigen is
either not exposed to reducing agents (e.g., Dithiothreitol (DTT)
or .beta.-mercaptoethanol) or not exposed to a sufficient amount of
reducing reagent to completely denature the antigen.
In one embodiment of the current invention, an antigen with
predetermined amount can be included as operational control to
ensure the consistency of experiment. The absolute level of the
antigen needs to be measured each time, and only when the measured
result is within the certain range of the pre-determined amount
before the overall experiment can be accepted as valid.
The QDB analysis can also be performed using the same lysate with
more than one antibody against the same biomarker. These antibodies
can direct against the same epitope, or against different epitopes
of the biomarker. A cutoff reference can be developed for these
antibodies respectively, and the sample can be determined either as
positive or negative in reference to these cutoff reference values.
Samples agreed by all antibodies can be accepted as positive or
negative, and those with disagreeable results can be assigned as
equivocal.
In one embodiment of current invention, the absolute levels of more
than one biomarker can be quantified using the same sample, and the
combination of these information together with relevant clinical
information, including, but not limited to, the disease free
survival, the overall survival, the hazard ratio, the side
response, the age, the progression stage of the disease, can be
used to find a pattern, and this pattern can be used for diagnosis
and prognosis purpose.
In one embodiment of current invention, the absolute level of a
biomarker can be measured with more than one antibody, and the
absolute levels of a biomarker based on each antibody respectively
over relevant clinical information, including, but not limited to,
the disease free survival, the overall survival, the side response,
the hazard ratio, the age, the progression stage of the disease,
can be used to find a pattern respectively, and individual pattern
from different antibodies can be used to provide antibody-specific
information for diagnosis and prognosis purpose.
Another aspect of the present invention relates to a reference
database for diagnosing cancer in a patient based on quantitative
analysis of a marker in a biopsy sample from the patient. The
reference database includes a plurality of reference profiles. each
of the plurality of the reference profile is prepared by steps of:
(a) providing a biopsy sample from a cancer patient with a known
diagnosis; (b) measuring said marker with dot blot analysis for the
biopsy sample, wherein the quantitation result is an absolute
amount of said marker in the biopsy sample, normalized by the
biopsy sample volume or by the biopsy sample weight; and (c)
associating the quantitation result with the known diagnosis of the
cancer patient thereby obtaining a reference profile.
The step (b) described above can include (b1) incubating the biopsy
sample with a solution containing a binding agent that specifically
binding to said marker; and (b2) measuring said marker through QDB
analysis of the binding agent.
In the above reference database, preferably the marker is a protein
marker and the binding agent is an antibody. Advantageously, the
reference database is stored in a computational memory chip.
Yet another aspect of the present invention is directed at a method
for diagnosing cancer in a patient. The method includes steps of
(i) providing a reference database described above, (ii) obtaining
a biopsy sample from the patient; (iii) measuring the marker used
in the reference database in the biopsy sample from patient with
dot blot analysis, the measured result being an absolute amount of
the marker in the biopsy sample; (iv) comparing the measured result
of the marker with that of each reference profile stored in the
reference database; and (v) identifying a reference profile in the
reference database that has the best match and outputting the known
diagnosis associated with the identified reference profile.
In the above method, the cancer being diagnosed is solid tumor.
Still another aspect of the present invention relates to an
apparatus for diagnosing cancer in a patient. The apparatus
contains the reference database just described.
Yet another aspect of the present invention relates to a kit for
diagnosing cancer in a patient. The kit contains the above
reference database. Alternatively, the kit contains a unit
configured to access online the above reference database.
The details of the invention are set forth in the drawing and the
description below. Other features, objects, and advantages of the
invention will be apparent to those persons skilled in the art upon
reading the drawing and the description, as well as from the
appended claims.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A-1D: Measuring the absolute her2 level with EP3 antibody
using dot blot analysis. Purified her2 protein was used to define a
dose curve, and the captured image was converted into number using
ImageQuant from Li-Cor. A dose curve was established, and used to
convert the captured dot blot image from patient samples into
absolute Her2 level among these patients.
FIGS. 2A-2H defining the linear range of three known antibodies for
IHC analyses of Her2, Ki67 and ER in QDB analysis. Recombinant
proteins of Her2, Ki67 and ER were used as protein standard. The
dose response curves were established using QDB analyses with these
antibodies respectively.
FIGS. 3A-3D show the absolute levels of Her2, Ki67, ER in breast
cancer tissues extracted from FFPE specimens, measured with QDB
analysis, and the absolute Her2 levels in frozen stomach tissues.
The result was further grouped based on their IHC results
respectively.
FIGS. 4A-4D show how to define a cutoff value to convert continuous
data into categorized results based on Her2 absolute levels
measured with QDB analysis, and the corresponding IHC results,
obtained from local hospital.
FIG. 5 shows how to analyze the clinical data using absolute value
of a biomarker. In this case, the potential link between Her2
absolute level and the histologic grade of breast cancer patients
was investigated.
DETAILED DESCRIPTION
Before the present methods are described, it is to be understood
that this invention is not limited to particular method described,
as such may, of course, vary. It is also to be understood that the
terminology used herein is for the purpose of describing particular
embodiments only, and is not intended to be limiting, since the
scope of the present invention will be limited only by the appended
claims.
Unless otherwise defined in this disclosure, all technical and
scientific terms used herein have the same meaning as is commonly
understood by one of skills in the art to which this disclosure
belongs.
The subject methods are useful primarily for diagnostic purposes.
Thus, as used herein, the terms "determining," "measuring," and
"assessing," and "assaying" are used interchangeably and include
both quantitative and qualitative determinations. These terms can
also refer to both quantitative and semi-quantitative
determinations and as such, the term "determining" is used
interchangeably herein with "assaying," "measuring," and the like.
Where a quantitative determination is intended, the phrase
"determining an amount" of an analyte and the like is used. Where
either a quantitative and semi-quantitative determination is
intended, the phrase "determining a level" of an analyte or
"detecting" an analyte is used.
Whenever applicable, "quantitative" assays in general provide
information on the amount of an analyte in a sample relative to a
reference (control), and are usually reported continuously
numerical, where a "zero" value can be assigned where the analyte
is below the limit of detection. "Semi-quantitative" assays
yielding an approximation of the quantity or amount of a substance;
between a qualitative and a quantitative result, where a "zero"
value can be assigned where the analyte is below the limit of
detection.
The term "diagnosis" is used herein to refer to the identification
of a molecular or pathological state, disease or condition, such as
the identification of a molecular subtype of breast cancer or other
type of cancer.
The term "prognosis" is used herein to refer to the prediction of
the likelihood of cancer-attributable death or progression,
including recurrence, metastatic spread, and drug resistance, of a
neoplastic disease, such as breast cancer.
The term "predicative" is used here to refer to the prediction of
the outcome of therapeutic intervention.
The terms "subject," "host," "patient," and "individual" are used
interchangeably herein to refer to any mammalian subject for whom
diagnosis or therapy is desired, particularly humans.
As used herein, the term "unit," in the context of an
antibody-based essay, refers to an epitope of a target protein. In
a more general setting, "unit" could represent a hybridizing region
recognized by a binding agent.
Quantitation by QDB
The quantitatively measurement of the protein expression level of a
biomarker can be achieved at tissue level using immunoblot method.
The immunoblot method is to be considered to its broadest context.
As long as the analytical process is antibody-based, and a solid
phase is involved in the process, it can be considered as
immunoblot analysis. The solid phase includes, but not limited to,
a membrane, the ELISA plate, the QDB plate (AKA, the plate used in
QDB analysis).
There is no limitation to how the antibody is validated. The
antibody can be validated using Western blot analysis when linear
epitope is used; it can also be validated based on prior
experience. For example, a lot of antibodies have been used in
clinical diagnosis and prognosis. These antibodies include, but not
limited to ASR or IVD antibodies, and other clinically accepted
antibody for immunohistochemistry or flow cytometry analysis. All
these antibodies can be used in QDB analysis without further
validation.
A detection antibody can also be validated in reference to a known
detection antibody. In one embodiment of current invention, a
detection antibody is used to analyze more than one sample, and the
results are compared with those from a known antibody. For example,
4B5 clone from Roche for HER2 protein. When the concordance is
beyond certain value, this antibody can be considered as a
validated antibody.
In one embodiment of current invention, the expression level of a
biomarker is measured with a typical immunoblot method. This method
includes, but not limited to two features, including 1) a validated
antibody and 2), A dose curve can be established using a protein
standard.
In one embodiment of current invention, the expression level of a
biomarker is measured with a typical immunoblot method. This method
includes, but not limited to three features, including 1) a
validated antibody and 2) a dose curve can be established using a
protein standard, and 3) the immunocomplex on the membrane is
individual quantified, without the interference from neighboring
unit during the detection process.
In one embodiment of current invention, a reference sample can be
included in the experiment. The reference sample is processed with
a sample of interest in the same immunoblot analysis. The result of
the reference sample is compared with that of sample of interest to
determine if the sample of interest is positive when the result of
sample of interest is above that of reference sample, or vice
versa.
In another embodiment of current invention, more than one reference
sample are included in the experiment. The reference samples are
processed together with sample of interest in the same immunoblot
analysis. The results of reference samples are used to define a
ranked order, and the result of sample of interest is determined in
reference to those of reference samples. For example, if reference
sample 1 and reference sample 2 are included to define the upper
limit of 1+ and 2+, and the result from sample of interest is
between those of reference sample 1+ and reference sample 2+, the
sample of interest is assigned as 2+.
In one embodiment of current invention, the expression level of a
biomarker is measured quantitatively using dot blot method. The Dot
blot analysis includes, but not limited to, features including a) a
validated antibody; b), defined linear range when the antibody is
used to analyze one type of sample, and c), the immunocomplex on
the membrane is detected as an image, and d), the conversion of
image into a number.
In another embodiment of current invention, the expression level of
a biomarker is measured quantitatively using QDB method. The QDB
analysis includes, but not limited to, features including a) a
validated antibody; b), defined linear range when the antibody is
used to analyze one type of sample; and c), the immunocomplex on
the membrane is individual quantified, without the interference
from neighboring unit during the detection process.
The categorized results in current prevailing form of IHC should be
considered in its broadest context. These results can be binary
(positive or negative), or ranked (0, 1+, 2+, 3+ or strong, medium
or weak), or by percentage, or in combination of all the above.
Several scoring systems for IHC, including H score, is still
considered as categorized results, as the result is discrete in
nature
The result from the method disclosed in this invention is a
continuous data. Although this data can be converted into a
categorized result as in prevailing IHC system to facilitate the
comparison and understanding of the clinical significance of the
result from this invention, this conversion is based on a cutoff
reference derived from mathematical analysis, and the category
where it belongs to can vary in reference to difference cutoff
reference. In one embodiment of current invention, the measured
Her2 levels in breast cancer tissue can be used for statistical
analysis, and a cutoff value can be derived from Receiver Operative
Characteristic analysis (ROC) based on IHC results, and the
absolute level in breast cancer tissue can be assigned as either
"positive" or "negative" in reference to the derived cutoff value
from ROC analysis, with absolute Her2 level above the cutoff value
as positive, and verse versa.
Absolute Measurement
The result from current invention can be relative, or, in
combination with a protein standard, to be absolute. The terms
"relative" and "absolute," referring to two ways to take a
measurement, should be taken into their broadest context. While
relative measurement is measuring one thing compared to another
thing, absolute measurement is measuring things in known amounts
with standard units. Perhaps, the most significant difference
between these two measurements lies in each's applicable scope. A
relative result is only meaningful under the same experimental
setting, while an absolute result should be comparable across a
number of different analyses, even analyses taken at vastly
separate places or times.
Accordingly, the absolute measurement used herein is achieved with
the inclusion of a protein standard. This protein standard should
be taken into its broadest context. It refers to an antigen of
pre-determined amount, or one antigen of more than one
pre-determined amount. It includes, but not limited to, the amount
pre-determined by weight, by the percentage, by the volume, by the
number of molecules (for example, the mole), or a combination of
any two or more of above. It can be in pure form, or it can be in a
mixture. It can be in native form, or it can be in non-denatured
form, or it is in denatured form. In this disclosure, the
non-denatured form can be considered as any form between native
form and denatured form.
Biomarkers
A "sample" or "patient sample" or "biological sample," which is
used interchangeably herein, generally refers to a sample which may
be tested for a particular molecule, preferably a specific marker
molecule associated with a biological signature, such as a
biomarker shown in the paragraph below. Samples may include, but
are not limited to, peripheral blood cells, CNS fluids, serum,
plasma, buccal swabs, urine, saliva, tears, pleural fluid and the
like. A sample used in the present invention generally refers to a
tissue.
The term "marker" or "biomarker" here is to be defined at its
broadest context. A "marker" or "biomarker," which is used
interchangeably herein, generally refer to an organic biomolecule
(e.g., a polypeptide) which is differentially present in a sample
taken from a subject of one phenotypic status (e.g., having a
disease) as compared with that from another phenotypic status
(e.g., not having the disease or having a different disease). A
biomarker is thus often established if differentially present
between two different phenotypic statuses, when the mean or median
level of the biomarker in a first phenotypic status relative to a
second phenotypic status is calculated to represent statistically
significant differences.
In the present invention, the biomarker is protein molecule
measurable related with a biological or a disease state. It can be
well established diagnostic biomarkers (for example, a diagnostic
biomarker for IHC), or it can be biomarkers newly identified for in
vitro diagnostics.
In one embodiment of present invention, the biomarker is used for
diagnosis of solid tumor.
The known biomarkers for diagnostics include, but not limited to
ACTH, ACTIN, ADENOVIRUS, AFP, ALK-1, AMYLOID A, ANDROGEN RECEPTOR,
ANNEXIN, ARGINASE-1, BAP1, B-AMYLOID, BCL-1, BCL-2, BCL-6, BEREP4,
Beta-Catenin, BOB1, BRACHYURY, BRST-2, C3d, C4d, CALCITONIN,
CALDESMON, CALPONIN, CALRETININ, CD14, CD117, CD117, BM, CD123,
CD138, CD15, CD163, CD1a, CD2, CD20, CD21, CD23, CD25, CD3, CD30,
CD31, CD33, CD34, CD34, BM, CD38, CD4, CD43, CD45RA, CD45RB,
CD45RO, CD5, CD56, CD57, CD61, CD68, CD7, CD79a, CD8, CD99, CDX2,
CEAm, CEAp, Chromogranin, Chymotrypsin, Claudin 3, Claudin 4, CK
MIX, CK20, CK34BE12, CK5/6, CK7, CK19, CKAE1/AE3, CKCAM5.2, CMV,
c-Myc, CXCL13, CYCLIN D3, D2-40, DBA-44, Desmin, DOG-1, EBV, LMP,
E-Cadherin, EGFR, EMA, EMA-Perineurioma, ER, ERG, Factor 13a,
Factor 8, FOXP1, FSH, GALECTIN-3, Gastrin, GATA-3, GFAP, GH,
Glucagon, Glut1, Glutamine synthetase, Glypican-3, GPC, GRANZYME,
H. pylori, HBC, HBS, hCG, HepPar 1, Her2neu, HGAL, HHV-8, HMB-45,
HPL, HSV, IDH1, IgA, IgD, IgG, IgG4, IgM, Inhibin, INI, Insulin,
ISH EBV, ISH KAPPA, ISH LAMBDA, KAPPA, KBA62, KI67, Lambda,
LANGERIN, LAT, LEF1, LH, LM02, LYSOZYME, MAP-2, MCT, MELAN A, MITF,
MLH1, MNDA, MOC-31, MPO, MSH2, MSH6, MUC2, MUC5AC, MUM1, Myogenin,
Napsin A, NB84, NEU N, Neurofilament, NKI/C3, NKX3.1, NPM, NSE,
NUT, 2-Oct, Oct-3/4, p16, p53, p57, p63, Parvovirus, PAX-2, PAX-5,
PAX8, PCSK9, PD-1, Perforin, PHH3, PHLDA1, PIN4, PLAP, PMS2, PR,
PRAP, Prolactin, Prox1, PSA, RNA, S100, S100P, SALL4, SF-1, SMA,
SMMS, Somatostatin, SOX-10, SOX11, SCAP, S1P, SREBP, Spirochetes,
STATE, SV40, SYNAP, Tamm-Horsfall, T-bet, TCL-1, TCR, TCR, Gamma,
TdT, THYRO, TIA-1, TOXO, Transthyretin, TRAP, TSH, TTF1,
Tyrosinase, Vimentin, WT1, WT1 (C-19), ZAP70.
This method can be adopted to analyze the antigen in fresh tissue,
or it could be used to analyze antigen in formalin fixed paraffin
embedded block (FFPE). The antigen can be extracted from the tissue
or FFPE by any method as long as the antigen from extraction
process allows the process of antibody-antigen interaction to
happen. For antigen in FFPE block, the slice needs to be
de-paraffined with and without the antigen retrieval step to
facilitate antigen-antibody interaction.
REFERENCE DATABASE
The QDB results from more than one sample of interest, together
with their matching clinical information, can be used to setup a
database for mathematical analysis (AKA, QDB database). A reference
database can thus be built for diagnostic purposes.
The terms "reference" and "control" are used interchangeably to
refer to a known value or set of known values against which an
observed value may be compared. The known value represents an
understood correlation between two parameters, e.g., a level of
expression of a marker and its associated phenotype. As used
herein, the known value constitute a reference profile in a
reference database.
Accordingly, a reference database can be prepared storing a number
of reference profiles for diagnostic purpose, each recording a
marker expression level of a sample obtained from a subject with
either a known diagnosis or known clinical outcome after
therapy.
In one embodiment, the present invention also includes a method of
determining a patient profile that best matches one or more
reference profiles in a reference database. The method includes
steps of (a) comparing, on a suitably programmed computer, the
level of expression of a marker in a sample from a patient with
reference profiles in a reference database to determine a measure
of similarity between the patient profile and each of the reference
profiles; (b) identifying, on a suitably programmed computer, a
reference profile in a reference database that best matches the
patient profile based on a maximum similarity among the measures of
similarity determined in step (a); and (c) outputting to a user
interface device, a computer readable storage medium, or a local or
remote computer system; or displaying, the maximum similarity or
the associated phenotype of the reference profile in the reference
database that best matches the patient profile.
Absolute Value-Based Diagnostic Method
The putative association between the expression level of a
biomarker and a clinical trait is explored using a mathematical
method to analyze the information from a QDB database. The examples
include, but not limited to, the relative expression level of HER2
with the disease free survival of the patient. The predicative
value of Ki67 in treating stomach cancer patients. This information
may provide prognosis for other patient in the same analysis.
As pointed out above, diagnostic tests used in clinical practice
often rely on a single analyte, and therefore do not capture the
value of its potentially intrinsic correlations with other
parameters, including some clinical readings. Moreover, diagnostic
tests are frequently not quantitative, relying on
immunohistochemistry, which often yields different results in
different laboratories, in part because the reagents are not
standardized, and in part because the interpretations are
subjective and cannot be easily quantified.
When the protein standard is included in the QDB analysis, the
absolute level of a biomarker can be achieved, greatly facilitating
benchmarking across different clinical laboratories. The QDB
results from more than one sample of interest, together with their
matching clinical information, can be used to setup a QDB database
for mathematical analysis. The putative association between the
absolute level of a biomarker and a clinical trait is explored
using a mathematical method. The examples include, but not limited
to, the absolute expression level of HER2 with the disease free
survival of the patient. This information may provide diagnosis and
prognosis for a patient in the future when the absolute level of
the biomarker is measured.
The absolute results from more than one QDB analysis can be
combined together to increase the size of the database for
mathematical analysis. The putative association between the
absolute level of a biomarker and a clinical trait is explored
using a mathematical method. The examples include, but not limited
to, the absolute expression level of HER2 with the disease free
survival of the patient, or the predicative value of Ki67 in
treating stomach cancer patient. This information may provide
prognosis and diagnosis for a patient with known absolute level of
a biomarker.
The absolute level of more than one biomarker can be measured using
a sample of interest. These results, together with the clinical
information of the patient of interest, can be used to setup a QDB
reference database for mathematical analysis to provide diagnosis
and prognosis to a patient when the absolute levels of these
biomarkers are measured. Reference profiles can be obtained, each
carrying the absolute level of biomarker for a patience with either
a known diagnosis or known clinical outcome after therapy. Later
for a new patient, his or her absolute level of the biomarkers can
be determined and compared with those in the reference database. A
match with the absolute level of a stored reference profile can be
indicative of the associated diagnosis or prognosis for therapy for
the new patient.
The clinical trait is to be considered in its broadest context. The
trait may include, but not limited to, age, sex, blood pressure,
glucose level, cancer stage, hazard ratio, disease free survival,
or any information relevant to the diagnosis, prevention, treatment
of the patient.
Another substantial advantage comes from the diagnostic method of
the present invention. Specifically, the method can also be
utilized for screening patients prior to entry into a clinical
trial. A major impediment to cancer therapeutic trials aimed at
personalized treatment is the lack of biomarkers available for
stratifying the patients. The validation of an absolute value-based
screening tool for cancer patients could significantly reduce the
costs of such trials by using a single standard and refining the
study entry process. With reduced costs for identification and
screening of patients, the new methods for screening of the present
invention facilitate recruitment, screening, and/or selection of
patients from a broader range of populations and/or clinic
settings, thereby offering underserved patient populations the
opportunity to engage in clinical trials, which has been a major
limitation to the majority of previously conducted trials in
oncology.
Moreover, today's oncologists frequently have a number of treatment
options available to them, including those labeled as "standard of
care," and other drugs that do not carry such a label yet exhibited
efficacy in certain types of cancer patients. Likelihood of good
treatment outcome can be maximized if patients could be assigned to
their optimal cancer treatment options as quickly as possible
following diagnosis. The method of the present invention fulfills
these needs by providing an enabling tool.
The absolute level of a biomarker can be measured with QDB
analysis, or it may be measured with other method in addition to
QDB analysis. In one embodiment of current invention, the absolute
level of a sample of interest is verified with QDB analysis.
The absolute level of a biomarker for setting up a QDB database can
be measured with QDB analysis, or it can be verified with QDB
analysis. A QDB database should be considered in its broadest
context. A QDB database includes at least one sample of interest
with the absolute level of one biomarker measured or verified with
QDB analysis.
The absolute level of a biomarker can be measured using more than
one antibody. These antibodies can be against the same epitope, or
they can be against different epitope of the biomarker. The
absolute levels from these antibodies may be the same, or they are
different from each other. When the absolute levels are different
from each other using different antibodies, they should be
specified with the antibody used, and be processed individually for
diagnosis and prognosis purpose.
In one embodiment of current invention, when more than one
detection antibody are used to measure the absolute level of a
biomarker in a sample, If the diagnosis or prognosis is the same
from these antibodies, the diagnosis or prognosis is considered as
valid. If the diagnosis or prognosis is in disagreement, the
diagnosis or prognosis is considered as "equivocal", which requires
further verification.
Any antibody used for antigen-antibody interaction can be used for
QDB analysis as long as it is validated as specific to the antigen
of interest. In one embodiment of current invention, the antibody
is monoclonal. In yet another embodiment of current invention, the
antibody is polyclonal.
In one embodiment of current invention, an antibody for
immunohistochemistry can be used for QDB analysis using tissue or a
cell from a human or an animal.
In one embodiment of current invention, an antibody for flow
cytometry can be used for QDB analysis using tissue or a cell from
a human or an animal.
In yet another embodiment of current invention, an antibody for
antigen-antibody based assay can be used for QDB analysis using
tissue or a cell from a human or an animal.
In one embodiment of current invention, an antibody for a biomarker
for QDB analysis of a sample can be labeled with fluorescence dye
detectable at one wavelength, while another antibody for another
biomarker for QDB analysis of the same sample can be labeled with
fluorescence day detectable at another wavelength can be used
simultaneously to analyze both biomarkers in the same sample.
In another embodiment of current invention, an antibody for a
biomarker for QDB analysis of a sample can be labeled with one
isotope, while another antibody for another biomarker for QDB
analysis of the same sample can be labeled with another isotope can
be used simultaneously to analyze both biomarkers in the same
sample. In yet another embodiment of current invention, an antibody
for a biomarker for QDB analysis of a sample can be labeled in one
method, for example, with Horseradish peroxide (HRP); while another
antibody for another biomarker for QDB analysis of the same sample
can be labeled with a florescent dye to allow simultaneously
measurement of two biomarkers in the same sample.
As used herein "reporter enzyme" is to be taken in its broadest
context. A reporter enzyme can be any modification of the antibody
in immunodetection assay with the purpose to aid the detection of
the antibody. For example, a report enzyme can be, but not limited
to, antibody directly labeled with radioactive isotope like Iodide
125, or reporter enzymes like alkaline phosphatase or horseradish
peroxidase, or with a fluorescence day. The detection of the amount
of reporter enzymes associated with antibody is through the
formation of a detectable product as the readout of the amount of
reporter enzymes in the detection reaction. The product can be
radioactive, luminescent, fluorescent, or a product with
characteristic absorbance or reflection spectrum in the visible or
outside the visible range. When a complement is used to detect the
bound antigen-antibody complex, it may either be labeled in any one
of the above ways, or be detected in turn by a specific
anti-complement antibody.
A report enzyme can be, but not limited to, antibody indirectly
labeled with radioactive isotope like Iodide 125, or reporter
enzymes like alkaline phosphatase or horseradish peroxide. Antibody
can be, but not limited to, indirectly labeled through a secondary
antibody, and the secondary antibody is directly or indirectly
labeled with radioactive isotope like Iodide 125, or reporter
enzymes like alkaline phosphatase or horseradish peroxide. In one
embodiment, the secondary antibody is labeled with biotin, and
indirectly further labeled with a horseradish peroxide through a
streptavidin molecule.
As used herein "antigen" and an "antibody" are to be taken in their
broadest context. An "antigen" can be a molecule, a cell, a virus,
or a particle. The term "antigen" may be used to refer to a
chemical compound, a peptide, a protein, an RNA, a DNA, a cell
(proteins released in situ), or a virus particle (proteins released
in situ) or any molecules that may evoke the production of one or
more antibodies by a host animal, including human. An antigen may
also be a product comprising any two or more of the molecules or
moieties crosslinked together. An antigen can exists either in a
pure form, or it can exist in a mixture. An antigen can be in a
modified form (e.g., modified by a chemicals) or be in an
unmodified form.
Reference herein to an "antibody" is to be taken in its broadest
context. An "antibody" is a polypeptide that binds to "an antigen".
An antibody includes, but is not limited to, a traditional
antibody, a fragment of a traditional antibody containing an
antigen binding site, a recombinant antibody containing an antigen
binding site, a protein which binds to an antigen, and a product
that comprises of crosslinking any two or more of the above. An
antibody can exist either in a pure form, or in a mixture. An
antibody can be in a modified form (e.g., modified by a chemical)
or be an unmodified form.
"Solid tumor" is to be considered in its broadest term. A solid
tumor refers to any tissue, cancer or non-cancer, which is
connected at cellular level, and not existing as a single cell. It
includes, but not limited to, breast cancer, prostate cancer,
bladder cancer, stomach cancer, kidney cancer, Melanoma of skin,
Cervix uteri cancer, head and neck cancer, brain cancer, thyroid
cancer lung cancer, liver cancer, pancreatic cancer, and colorectal
cancer.
It is to be understood that the exemplary embodiments described
herein are that for presently preferred embodiments and thus should
be considered in a descriptive sense only and not for purposes of
limitation. Descriptions of features or aspects within each
embodiment should typically be considered as available for other
similar features or aspects in other embodiments.
Further, Examples put forth are not intended to represent that the
experiments below are all or the only experiments performed.
Efforts have been made to ensure accuracy with respect to numbers
used (e.g., amounts, temperature, etc.) but some experimental
errors and deviations should be accounted for.
Materials and Methods
Human Subjects and Human Cell Lines
Both Frozen tissues and formalin fixed paraffin embedded (FFPE)
slices were obtained from local hospitals together with their
clinical information including IHC scores (0, 1+, 2+ or 3+). Some
of them were with FISH results. MCF-7 and BT-474 cell lines were
purchased from the Cell Bank of Chinese Academy of Sciences
(Shanghai, China), and lysates prepared from these two cell lines
were used as HER2 negative and positive controls.
General Reagents.
All general reagents used for cell culture were purchased from
Thermo Fisher Scientifics (Waltham, Mass., USA) including cell
culture medium and culture dishes. The protease inhibitors were
purchased from Sigma Aldrich (St. Louis, Mo., USA). All other
chemicals were purchased from Sinopharm Chemicals (Beijing, P. R.
China). Recombinant human HER2/ErbB2/CD340 (676-1255) protein was
purchased from Sino Biological Inc. (Beijing, China). QDB plate was
manufactured by Yantai Zestern Biotechnique Co. Ltd at Yantai,
China.
Ventana anti-HER2/neu(4B5) rabbit monoclonal primary antibody and
anti-Human estrogen receptor (clone Sp1) were purchased from Roche
Diagnostics GmbH. Rabbit anti-HER2 antibody (clone EP3) and Mouse
anti-human Ki67 (clone MIB1) were purchased from ZSGB-BIO (Beijing,
China). HRP labeled Donkey Anti-Rabbit IgG secondary antibody was
purchased from Jackson Immunoresearch lab (Pike West Grove, Pa.,
USA).
Preparation of Cell and Tissue Lysates
For frozen tissues, about 150 mg tissues were cut from tissue
biopsies, and processed in 300 .mu.l lysis buffer (50 mM HEPES, 137
mM NaCl, 5 mM EDTA, 1 mM MgCl, 10 mM Na.sub.2P.sub.2O.sub.7, 1%
TritonX-100, 10% glycerol) with protease inhibitors (2 .mu.g/ml
Leupeptin, 2 .mu.g/ml Aprotinin, 1 .mu.g/ml pepstatin, 2 mM PMSF, 2
mM NaF) with a handheld tissue homogenizer for 30 s before they
were centrifuged at 12000.times.g for 5 mins. The supernatants were
collected for immunoblot analysis. The total protein concentration
was measured using Pierce BCA protein assay kit in accordance to
the manufacturer's instructions. To prepare lysates from MCF-7 and
BT-474 cells, cells were lysed in lysis buffer with protease
inhibitors by pipetting up and down for 50 times. Supernatants were
collected after centrifugation and the total protein concentration
was determined by the BCA protein assay kit.
For Formalin fixed paraffin embedded (FFPE) blocks, two 2.times.15
.mu.m slices were collected, de-paraffined and processed in 3000
lysis buffer (50 mM HEPES, 137 mM NaCl, 5 mM EDTA, 1 mM MgCl, 10 mM
Na.sub.2P.sub.2O.sub.7, 1% TritonX-100, 10% glycerol) with protease
inhibitors (2 .mu.g/ml Leupeptin, 2 .mu.g/ml Aprotinin, 1 .mu.g/ml
pepstatin, 2 mM PMSF, 2 mM NaF) before they were centrifuged at
12000.times.g for 5 mins. The supernatants were collected for
immunoblot analysis. The total protein concentration was measured
using Pierce BCA protein assay kit in accordance to the
manufacturer's instructions. To prepare lysates from MCF-7 and
BT-474 cells, cells were lysed in lysis buffer with protease
inhibitors by pipetting up and down for 50 times. Supernatants were
collected after centrifugation and the total protein concentration
was determined by the BCA protein assay kit.
Dot Blot Analysis
Purified her2 protein was serially diluted as indicated in the
figure, and spotted on the a membrane together with total proteins
extracted from FFPE slices. These samples were processed in a
typical dot blot process until the image was captured using C-Digit
Western blot scanner from Li-Cor. The image was converted into a
number using ImageQuanti program from Li-Cor, and a protein
standard curve was established to convert the images captures from
patient samples into absolute biomarker levels.
QDB Analysis
The linear range of a specific antibody (EP3 or 4B5 clone for Her2;
MIB1 for Ki67, SP1 for Estrogen receptor (ER)) was determined by
using a pooled lysate from patients testing positive respectively
for these biomarkers. The lysates were prepared first by mixing in
equal amount tissue lysates prepared from 3 to 4 breast or stomach
cancer tissues. The pooled lysates were serially diluted from 0-2
.mu.g to define the linear range of QDB analysis. A protein
standard either obtained commercially or expressed and purified in
the company was also serially diluted from 0-500 pg, and used to
define the linear range of QDB analysis.
The samples were applied onto the QDB plates at 20/unit in
triplicate, and were processed as described previous.sup.15. A
primary antibody was used for primary antibody incubation at 100
ul/well overnight at 4.degree. C. and a donkey anti-rabbit or
donkey anti-mouse secondary antibody was incubated with the plate
for 4 hours at room temperature. The plates were briefly rinsed
twice with TBST, and washed 5.times.10 mins before they were
inserted into a white 96-well plate pre-filled with ECL solution
prepared according to the manufacturer's instruction at 1000/well
for 3 mins. The chemiluminescence signal from individual well of
the plate was quantified by using the Tecan Infiniti 200 pro
Microplate reader with the option "plate with cover".
A linear regression formula was established using serially diluted
protein standard respectively. The formula was used to calculate
the total HER2 level in each breast cancer tissue samples, and the
results were corrected by total protein amount measured by the BCA
protein determination kit. To ensure the consistency of the
experiments, two cell lysates prepared from BT474 and MCF7 were
used as positive and negative controls. The lysates were prepared
in small aliquots. The absolute biomarker levels from two cell
lysates were measured with QDB analysis and documented prior to
being included and processed as regular samples in all the QDB
experiments performed. Samples with chemiluminescence reading less
than 2 fold over blank were regarded as non-detectable, and entered
as 0 for data analysis. The final result was further corrected by
the percentage of the purity of the recombinant HER2 protein.
Statistical Analysis
Common tests for statistical significance include, among others,
t-test, ANOVA, Kruskal-Wallis, Wilcoxon, Mann-Whitney and odds
ratio. Biomarkers, alone or in combination, provide measures of
relative likelihood that a subject belongs to a phenotypic status
of interest. As such, biomarkers can find use as markers for, for
example, disease, therapeutic effectiveness of a drug, and the
like. Biomarkers are thus analytes in assays that facilitate
diagnosis and the like.
All the data were presented as Mean.+-.Standard Deviation (SD). The
difference between individual groups was calculated using Microsoft
Excel using unpaired two-tailed Student's t tests. P value <0.05
was considered statistically significant. Receiver operating
characteristic (ROC) curves were generated using the GraphPad Prism
software version 7.0 (GraphPad Software Inc., USA), as indicated in
the figure legend.
EXAMPLES
Example 1 Teaches how to Measure Absolute Her2 Level in FFPE
Specimen Using Dot Blot Analysis
In 1A, recombinant Her2 protein was serially diluted and spotted
onto a membrane together with four patient samples extracted from
FFPE blocks.
The spotted protein standards and patient samples on the membrane
was processed in dot blot analysis using EP3 clone for Her2
protein, and the image was captured using C-Digit Western blot
scanner from Li-Cor, as shown in FIGS. 1A and 1B.
The image in FIG. 1A was converted into numbers using ImageQuant
software from Li-Cor, and these numbers were used to establish a
protein standard curve, as shown in FIG. 1C.
The images from 4 patient samples were also converted into number
using the same computer software, and the numbers were converted
into absolute Her2 level based on the protein standard curve
established in section [0098].
FIGS. 2A-2H teach how to define the linear range of the QDB
analysis using a specific antibody. FIG. 2A showed the linear range
of QDB analysis of frozen breast cancer tissues using Her2-EP3
antibody; FIG. 2B showed the linear range of QDB analysis of FFPE
slices using Her2-EP3 antibody; FIG. 2C showed the linear range of
QDB analysis of a recombinant Her2 protein, FIG. 2D showed the
linear range of QDB analysis of frozen stomach cancer tissue using
MIB1 antibody, FIG. 2E showed the linear range of QDB analysis of
FFPE slices using MIB1 antibody against Ki67, 2F showed the linear
range of QDB analysis of a recombinant fragment of Ki67 protein
using MIB1 antibody, FIG. 2G showed the linear range of QDB
analysis of a recombinant fragment of Estrogen Receptor (ER)
protein using SP1 antibody, and FIG. 2H showed the linear range of
QDB analysis of FFPE slices using SP1 antibody against ER.
FIGS. 3A-3D show the absolute level of Her2 (A), Ki67 (B) and ER
(C) in breast cancer tissues using Her2-4B5, MIB1 and SP2
antibodies respectively. In FIG. 3D, the Her2 absolute levels in
frozen stomach cancer were measured using Her2-EP3 antibody. The
results were average of three independent experiments in
triplicate, and were grouped based on their IHC scores.
FIGS. 4A-4D show how to convert a QDB result into a categorized
result using Receiving Operative Characteristic (ROC) analysis. The
results from QDB analysis were separated into negative and positive
groups based on their IHC results, and these two groups of numbers
were used to calculate the cutoff value with promising specificity
and sensitivity. FIGS. 4A and 4B showed ROC curves of QDB results
from HER2-EP3 and HER2-4B5 antibodies respectively. FIGS. 4C and 4D
showed how to separate QDB results into negative and positive
groups based on the suggested cutoff value from ROC analysis.
In FIG. 5, the absolute Her2 levels from 263 FFPE specimens were
measured, and plotted as a group based on the provided histologic
grade judged by local hospital based on Nottingham histologic
scores. There were 34 samples in group I, 125 samples with grade
II, and 104 samples in grade III group. The averages of three
grades were 0.08, 0.73, and 3.15 nmole/g. The her2 level were
significantly higher with patients in Grade III group than those
from Grade I and Grade II group, and absolute her2 level was
significantly higher among patients in Grade II group than those in
Grade I group. This result cannot be achieved with the current
prevailing categorized system with both IHC and FISH analyses.
All publications and patents cited in this specification are herein
incorporated by reference as if each individual publication or
patent were specifically and individually indicated to be
incorporated by reference and are incorporated herein by reference
to disclose and describe the methods and/or materials in connection
with which the publications are cited. The citation of any
publication is for its disclosure prior to the filing date and
should not be construed as an admission that the present invention
is not entitled to antedate such publication by virtue of prior
invention. Further, the dates of publication provided may be
different from the actual publication dates which may need to be
independently confirmed. To the extent a definition of a term set
out in a document incorporated herein by reference conflicts with
the definition of a term explicitly defined herein, the definition
set out herein controls.
It is believed that the following claims particularly point out
certain embodiments or combined thereof that are directed to one of
the disclosed inventions and are novel and non-obvious. Inventions
embodied in other combinations or subcombinations of features,
functions, elements and/or properties may be claimed through
amendment of the present claims or presentation of new claims in
this or a related application. Such amended or new claims, whether
they are directed to a different invention or directed to the same
invention, whether different, broader, narrower, or equal in scope
to the original claims, are also regarded as included within the
subject matter of the inventions of the present disclosure.
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